1. Field of the Invention
The present invention relates generally to the fields of molecular endocrinology and receptor pharmacology. More specifically, the present invention relates to a novel in vivo method for the identification of steroid hormone receptor agonists and antagonists.
2. Description of the Related Art
The ovarian hormones, estrogen and progesterone, are responsible, in part, for the regulation of the complex cellular events associated with differentiation, growth and functioning of female reproductive tissues. These hormones also play important roles in development and progression of malignancies of the reproductive endocrine system.
The biological activity of steroid hormones is mediated directly by a hormone and tissue-specific intracellular receptor. The physiologically inactive form of the receptor may exist as an oligomeric complex with proteins, such as heat-shock protein (hsp) 90, hsp70 and hsp56. Upon binding its cognate ligand, the receptor changes conformation and dissociates from the inhibitory heteroligomeric complex. Subsequent dimerization allows the receptor to bind to specific DNA sites in the regulatory region of target gene promotors. Following binding of the receptor to DNA, the hormone is responsible for mediating a second function that allows the receptor to interact specifically with the transcription apparatus. Displacement of additional inhibitory proteins and DNA-dependent phosphorylation may constitute the final steps in this activation pathway.
Cloning of several members of the steroid receptor superfamily has facilitated the reconstitution of hormone-dependent transcription in heterologous cell systems. Subsequently, in vivo and in vitro studies with mutant and chimeric receptors have demonstrated that steroid hormone receptors are modular proteins organized into structurally and functionally defined domains. A well defined 66 amino acid DNA binding domain (DBD) has been identified and studied in detail, using both genetic and biochemical approaches. The hormone binding domain (HBD), located in the carboxyl-terminal half of the receptor, consists of about 300 amino acids. It has not been amenable to detailed site-directed mutagenesis, since this domain appears to fold into a complex tertiary structure, creating a specific hydrophobic pocket which surrounds the effector molecule. This feature creates difficulty in distinguishing among amino acid residues that affect the overall structure of this domain from those involved in a direct contact with the ligand. The HBD also contains sequences responsible for receptor dimerization, hsp interactions and one of the two transactivation sequences of the receptor.
Although antiprogestins are presently used for pregnancy termination and in the treatment of hormone-dependent breast cancer, little is known about their mechanism of action. Nevertheless, certain insights to the role of ligand in progesterone receptor activation have come from the study of antihormones. Upon binding most antihormones, the receptor is driven to steroid responsive elements (SREs) within the regulatory regions of target genes. The affinity of antagonist activated receptor for DNA is indistinguishable from that of agonist-bound receptor. Nevertheless, in the presence of the antagonist, the receptor cannot activate transcription efficiently. A plausible explanation for this observation is that antihormones and hormones alter conformation differently. Agonists may induce conformational changes which expose the receptor transcription activation domain, whereas antagonists may promote DNA binding but induce a conformation which is transcriptionally silent.
Steroid receptors and other mammalian transcription regulators can function in yeast. This fact, coupled with the ease of genetic manipulation of yeast make it a useful system to study the mechanism of steroid hormone action.
A long felt need and desire in this art would be met by the development of methods to identify steroid hormone receptors agonists and antagonists. The development of such a method will facilitate the identification of novel therapeutic pharmaceuticals.